The formation of “hydrogen peroxide” in the reaction of oxyhemoglobin with methemoglobin-forming agents

doi:10.1016/0003-9861(57)90025-5

Archives of Biochemistry and Biophysics

Volume 71, Issue 1, September 1957, Pages 235-249

https://doi.org/10.1016/0003-9861(57)90025-5 Get rights and content

Abstract

1.
1. Comparisons were made among the reactions of phenylhydrazine, hydrazine, phenylhydroxylamine, and hydroxylamine with oxyhemoglobin and methemoglobin, especially with regard to the production of H₂O₂ or equivalent free radicals, methemoglobin production and reduction, and verdoglobin formation.
2.
2. Phenylhydrazine and phenylhydroxylamine were found to react with oxyhemoglobin and methemoglobin at a much faster rate than hydrazine or hydroxylamine.
3.
3. H₂O₂ or free radicals were produced rapidly by all the reducing agents mentioned, except hydrazine, when reacting with oxyhemoglobin, but the latter reacted too slowly to give a measurable amount of H₂O₂ or free radicals.
4.
4. Phenylhydroxylamine produced no measurable verdoglobin, although evidence is presented for free-radical production in this case.
5.
5. Possible mechanisms for verdoglobin production are discussed in light of the failure of phenylhydroxylamine to produce the green pigment.

References (23)

W. Heubner
Ergeb. Physiol
(1940)
G. Barkan et al.
Z. physiol. Chem
(1938)
R. Havemann
Biochem. Z
(1941)
F. Hoppe-Seyler
Zentr. med. Wiss
(1863)
R. Lemberg et al.
Biochem. J
(1941)
M. Kiese et al.
Biochem. Z
(1942)
A.B. Anderson et al.
J. Pathol. Bacteriol
(1934)
J.M. Neill et al.
J. Exptl. Med
(1924)
W.A. Hagan
J. Infectious Diseases
(1925)
S. Edlbacher et al.
Naturwissenschaften
(1937)
S. Edlbacher et al.
Naturwissenschaften
(1937)
S. Edlbacher et al.
Naturwissenschaften
(1937)

O. Warburg et al.

Biochem. Z

(1931)

Cited by (22)

Formation of free radicals and protein mixed disulfides in rat red cells exposed to dapsone hydroxylamine
1997, Free Radical Biology and Medicine
The hemolytic activity of dapsone is well known to reside in its N-hydroxylamine metabolites. Addition of dapsone hydroxylamine (DDS–NOH) to red cell suspensions causes damage such that when reintroduced into the circulation of isologous rats, the injured cells are rapidly removed by the spleen. Hemolytic activity is associated with the extensive formation of disulfide-linked hemoglobin adducts on red cell membrane skeletal proteins. To determine if free radicals could be involved in this process, rat red cells were incubated with DDS–NOH in the presence of the spin trap, 5,5′-dimethyl-1-pyrroline-N-oxide (DMPO) and subjected to EPR analysis. Addition of DDS–NOH (25–50 μM) to a red cell suspension gave rise to a four-line (1:2:2:1) EPR spectrum with coupling constants identical to those of a DMPO-hydroxyl radical adduct (DMPO–OH) standard. No other radicals were detected; however, preincubation of red cells with cysteamine caused the DDS–NOH-generated DMPO–OH signal to be replaced by a cysteamine thiyl radical adduct signal. DDS–NOH-treated red cells were also found to contain ferrylhemoglobin, indicating the presence of hydrogen peroxide. Furthermore, DDS–NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confirming the presence of oxygen radicals. These data support the hypothesis that oxygen radicals are involved in the mechanism underlying dapsone-induced hemolytic anemia. © 1997 Elsevier Science Inc.
Effects of phenylhydrazine on the electrophoretic behaviour of l-lactate dehydrogenase isozymes in Ictalurus species (Ictaluridae, Teleostei)
1993, Comparative Biochemistry and Physiology. Part C: Comparative
1. The effects of phenylhydrazine on LDH isozyme metabolism were studied in Ictalurus species.
2. Significant alterations were found and these varied for the different tissues studied.
3. The toxic effects of phenylhydrazine were linked to several factors: exposure time; hematocrit values, blood sugar levels; changes in normal metabolism with increased anaerobic glycolysis in some tissues; and energy recovery as a result of the fish's ability to react and its state of health.
4. Some hypotheses are made as to how phenylhydrazine may be dealt with: transformation, deposition and/or elimination.
Spectrin degradation in intact red blood cells by phenylhydrazine
1985, Biochemical Pharmacology
The effects of phenylhydrazine on intact red cells and on red cell ghost membrane proteins were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In intact red cells 1 mM phenylhydrazine induced a marked decrease in intensity of the α- and β-bands of spectrin without the formation of high molecular weight materials. Phenylhydrazine was also responsible for cross-linking of hemoglobin, which is apparent by the appearance of two new broad bands on the gel. Membrane glycoproteins were unaffected. Electrophoretic patterns of cytoskeletal proteins from phenylhydrazine-treated red cells obtained on two-dimensional SDS-polyacrylamide gels and stained with Coomassie blue or fluorescently labeled with monobromobirnane indicated the presence of a new band between bands 4.2 and 5 at 60–65 kilodaltons (K). An immunoelectrophoretic blotting procedure utilizing polyclonal IgG antibodies for α- and β-spectrin of the red cell cytoskeletal proteins revealed that the band observed at 60–65 K in the two-dimensional SDS-PAGE studies reacted with the antibodies. The presence or absence of glucose in the incubation medium and modification of oxyhemoglobin to met- or carboxyhemoglobin in the red cells did not protect the phenylhydrazine-mediated degradation of the major cytoskeletal proteins. Metal chelators and antioxidants had no effect on membrane protein changes. Ghost red cell proteins did not undergo changes at 1 mM phenylhydrazine in the presence or absence of hemoglobin, although at 5 mM phenylhydrazine the appearance of a faint high molecular weight band was observed. These results indicate that spectrin degradation without significant polymerization can be induced by phenylhydrazine.
A comparison between the ease of haemoglobin oxidation and modification by sulphide
1983, Comparative Biochemistry and Physiology -- Part B: Biochemistry and
- 1.
  1. The haemoglobin purified from a wide range of species undergoes facile sulphaemoglobin formation as well as oxidation.
- 2.
  2. Oxidation by either anions such as azide or organic electron transfer reagents shows similar facility.
- 3.
  3. Facile oxidation of a particular haemoglobin is associated with protection against sulphaemoglobin formation.
- 4.
  4. Oxidation and sulphaemoglobin formation in some instances occur via competing electron transfer reactions.
Membrane alterations in phenylhydrazine-induced reticulocytes
1980, Archives of Biochemistry and Biophysics
It is known that reticulocytes formed in animals in response to phenylhydrazine treatment have a shorter life span than those formed as a consequence of bleeding. The experiments presented illustrate that the reticulocytes formed consequent to the action of this drug exhibit membrane alterations, in the absence of intracellular oxidative changes (e.g., to hemoglobin), which might be expected to contribute to their decreased survival. These membrane alterations include the formation of flourescent chromolipids, a decrease in spectrin polypeptides, and an increase in high molecular weight membrane protein polymers. It is suggested that these effects are unique to the reticulocytes formed as a response to phenylhydrazine since they are a consequence of the peroxidation of membrane phospholipids initiated by this agent.
Cell death and free radicals: A mechanism for hydroxyurea teratogenesis
1979, Medical Hypotheses
Hydroxyurea is a drug which causes birth defects in a variety of animals. Its pharmacologic actions include rapid killing of proliferating cells and profound inhibition of the synthesis of DNA. The mechanism whereby hydroxyurea produces teratogenesis is unresolved although it is related to the pharmacologic actions. Careful reading of the scientific literature discloses several putative mechanisms for the teratogenic action of hydroxyurea, which account for the inhibited synthesis of DNA, but do not explain the $rapid$ onset of cell death. This paper presents a hypothesis that hydroxyurea causes rapid cell death through the initiation of uncontrolled free radical chain reactions. In vitro biochemical experiments have demonstrated the ability of compounds containing a hydroxylamine group (such as hydroxyurea) to form H₂O₂ and intermediate free radicals in biological fluids. Free radicals propagate themselves through chain reactions and can react swiftly and indiscriminately with the macromolecules of cells. Radical reactions are able to devastate cellular metabolism quickly by inactivating enzymes, cross-linking DNA, and altering membrane functions through lipid auto-oxidation. Preliminary experiments utilizing an antioxidant to counteract the free radical effects appear to support the hypothesis that the rapid cell death is caused by aberrant free radical reactions.

View all citing articles on Scopus

^☆: Work performed under contract No. W-7405-eng-26 for the U. S. Atomic Energy Commission.

²: Present address: Department of Physiology, Indiana University Medical School, Bloomington, Indiana.

View full text

The formation of “hydrogen peroxide” in the reaction of oxyhemoglobin with methemoglobin-forming agents☆

Abstract

Ergeb. Physiol

Z. physiol. Chem

Biochem. Z

Zentr. med. Wiss

Biochem. J

Biochem. Z

J. Pathol. Bacteriol

J. Exptl. Med

J. Infectious Diseases

Naturwissenschaften

Naturwissenschaften

Naturwissenschaften

Biochem. Z